Large-conductance Ca2+- and voltage-gated K+ (Slo1 BK) channels play numerous physiological and pathophysiological roles and their allosteric gating mechanism is subject to modulation by a variety of cellular signaling pathways. Increasing evidence suggests that certain lipids may serve as signaling molecules. We propose to reveal the biophysical and physicochemical mechanisms of modulation of Slo1 BK channels by two lipid messengers, phosphatidylinositol 4,5-bisphosphate (PIP2) and docosahexaenoic acid (DHA), an omega-3 long-chain polyunsaturated fatty acid enriched in oily fish. The effect of PIP2 on the Slo1 BK channel was reported recently but the mechanism is only poorly known. We will fill this critical knowledge gap by performing thorough mechanistic electrophysiological measurements. Furthermore, we will identify the structural determinants of the auxiliary beta subunit important for the action of PIP2. Our electrophysiological measurements will be complemented with measurements of tryptophan fluorescence of the isolate and purified Slo1 gating ring protein. The biophysical and physicochemical mechanisms of the action of DHA, an emerging lipid messenger, on the Slo1 channel will be also investigated similarly using the electrophysiological and fluorescence methods. The research outcome is expected to provide definitive mechanisms of the PIP and DHA actions on the allosteric gating mechanism of the Slo1 channel and establish the novel paradigm that the Slo1 channel is an omega-3 fatty acid receptor. The modulation of the Slo1 BK channel by DHA may underlie the health-promoting effects of omega-3 long-chain fatty acids.